CN105531290B - Method for obtaining APRIL-binding peptides, process for producing such peptides, APRIL-binding peptides obtained with said method/process and use of such peptides - Google Patents

Abstract

The present invention relates to a method for obtaining APRIL-binding peptides. APRIL-binding peptides can be obtained and/or selected using this method. Further aspects of the invention relate to cells comprising a nucleotide sequence encoding an APRIL-binding peptide of the invention, to processes for the production of APRIL-binding peptides and to APRIL-binding peptides obtained in said processes for production and/or selection. In view of the possible utility of the APRIL-binding peptides of the invention, further aspects of the invention relate to the diagnostic use of the APRIL-binding peptides of the invention.

Description

Method for obtaining APRIL-binding peptides, process for producing such peptides, APRIL-binding peptides obtained with said method/process and use of such peptides

Technical Field

The present invention relates to the fields of human and veterinary medicine, including medical/veterinary diagnostics and medical/veterinary research. More specifically, the invention relates to APRIL-binding peptides, including monoclonal antibodies, suitable for use in this or other fields.

Background

APRIL is expressed as a type II transmembrane protein, but unlike most other TNF family members, it is mainly processed as a secreted protein and cleaved in golgi organelles where it is cleaved by furin convertases to release soluble active forms (Lopez-Fraga et al, 2001, EMBO Rep 2: 945-51). In protein folding, APRIL assembles into non-covalently linked homotrimers with similar structural homology to many other TNF family ligands (Wallweber et al, 2004, Mol Biol 343, 283-90). APRIL binds two TNF receptors: b Cell Maturation Antigen (BCMA) and transmembrane activator-calcium modulator-cyclophilin ligand interactor (TACI) (reviewed in Kimberley et al, 2009, J Cell Physiol.218 (1): 1-8). Furthermore, APRIL has recently been shown to bind Heparan Sulfate Proteoglycans (HSPG) (Hendriks et al, 2005, Cell Death Differ 12, 637-48). APRIL has been shown to play a role in B Cell signaling and promotes proliferation and survival of human and murine B cells in vitro (reviewed in Kimberley et al, 2009, J Cell physiol.218 (1): 1-8).

APRIL is expressed primarily by subsets of immune cells, such as monocytes, macrophages, dendritic cells, neutrophils, B cells and T cells, many of which also express BAFF. In addition, APRIL may be expressed by non-immune cells, such as osteoclasts, epithelial cells, and various tumor tissues (reviewed in Kimberley et al, 2009, J Cell physiol.218 (1): 1-8). In fact, APRIL was originally identified based on its expression in cancer cells (Hahne et al, 1998, JExp Med 188, 1185-90). High expression levels of APRIL mRNA are found in a group of tumor cell lines as well as in human primary tumors such as colon and lymphoid cancers.

Retrospective studies of 95 Chronic Lymphocytic Leukemia (CLL) patients showed increased levels of APRIL in the serum, which is associated with disease progression and overall patient survival, with poorer prognosis in patients with high APRIL serum levels (Planelles et al, 2007, Haematologica 92, 1284-5). Likewise, APRIL is shown (increased levels) to be expressed in Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL) and Multiple Myeloma (MM) (reviewed in Kimberley et al, 2009, J Cell Physiol.218 (1): 1-8). Retrospective studies on DLBCL patients (NHL) showed that high APRIL expression in cancer foci was associated with poor survival (Schwaller et al, 2007, Blood 109, 331-8). Recently, serum levels of APRIL in the serum of patients with colorectal cancer were shown to have positive diagnostic value (Ding et al, 2013, Clin. biochemistry, http:// dx. doi. org/10.1016/j. clinbiochem.2013.06.008).

Due to its role in B-cell biology, APRIL also plays a role in many autoimmune diseases. Increased serum levels of APRIL have been reported in many SLE patients (Koyama et al, 2005, Ann Rheum Dis 64, 1065-7). Retrospective analysis revealed that APRIL serum levels tended to correlate with antibody titers against dsDNA. In addition, significantly increased levels of APRIL in synovial fluid were detected in patients with inflammatory Arthritis as compared to patients with non-inflammatory Arthritis (e.g., osteoarthritis) (Stohl et al, 2006, endo Metab Immune disease Drug Targets 6, 351-8; Tan et al, 2003, Arthritis Rheum 48, 982-92).

Several studies have focused on patients with a broader range of systemic immunity-based rheumatic diseases (now also including sjogren's syndrome: (

s syndrome), Reiter's syndrome, psoriatic arthritis, polymyositis and ankylosing spondylitis, and significantly increased levels of APRIL are found in these patients, which also indicates an important role for APRIL in these diseases (Jonsson et al, 1986, scanand JRheumatol supply 61, 166-9; roschke et al, 2002, J Immunol 169, 4314-21). Furthermore, increased APRIL serum levels were detected in sera from patients with atopic dermatitis (Matsushita et al, 2007, exp. In addition, serum APRIL levels are elevated in sepsis and the mortality of critically ill patients is predicted (Roderburg et al, j.critical Care, 2013, http:// dx.doi.org/10.1016/j.jcrc.2012.11.007). Finally, increased APRIL expression is also associated with Multiple Sclerosis (MS). Expression of APRIL was found to be increased in astrocytes of MS victims (sufferers) compared to normal controls. This is consistent with APRIL expression described in malignant gliomas and in the sera of patients with malignant gliomas (Deshayes et al, 2004, Oncogene23, 3005-12; Roth et al, 2001, Cell Death Differ 8, 403-10).

Summary of The Invention

Since APRIL is an important marker of diseases such as, but not limited to, autoimmune diseases, inflammatory diseases and malignancies, it is important to detect APRIL in the serum of human subjects. The currently available assays involve the use of polyclonal antibodies (undefined and limited availability) (plailles et al, 2007, haematologic 92, 1284-5), which do not reproduce the reported levels of APRIL in serum, and/or the quantification of APRIL is severely affected by the presence of human serum (BioLegend, see example 1), require an immunoglobulin adsorption step (Matsushita et al, 2007, exp. dermatology 17, 197-. In view of the shortcomings of the prior art anti-APRIL antibodies, the inventors of the present invention set out to develop methods for identifying and obtaining APRIL-binding peptides suitable for use in the context of detecting APRIL in a human sample, preferably a blood-derived sample (e.g., a serum sample). In particular, methods were designed and developed to select for antibody-expressing, rarely abundant B cells from APRIL-immunized mice.

Briefly, the method of the invention for obtaining an APRIL-binding peptide comprises the steps of:

-providing a library of binder peptides;

-selecting APRIL-binding peptides from said library by means of affinity selection using target peptides immobilized on a solid support, said target peptides comprising a plurality of APRIL epitopes and APRIL receptor binding regions of APRIL;

characterized in that the target peptide interacts with a peptide comprising the APRIL-binding region of the APRIL receptor or an APRIL-binding equivalent thereof, a shielding peptide (shielding peptide). In this way, APRIL-binding peptides can be obtained and/or selected.

The developed methods can be widely developed and used to obtain a wide range of APRIL-binding peptides, such as APRIL-binding antibodies.

Further aspects of the invention relate to APRIL-binding peptides obtained with the methods of the invention, cells comprising the nucleotide sequences encoding APRIL-binding peptides of the invention, processes for producing APRIL-binding peptides and APRIL-binding peptides obtained with such processes. Furthermore, the use of the APRIL-binding peptides of the invention in diagnostic tests, preferably in ex vivo diagnostic tests, is also within the scope of the invention.

Brief description of the sequences

The sequences presented in the sequence Listing relate to the V of five immunoglobulins (hAPRIL.130, hAPRIL.132, hAPRIL.133, hAPRIL.135, hAPRIL.138) obtained by the method of the invention_HAnd V_LThe amino acid sequence of the strand and the coding DNA sequence. Furthermore, V of these immunoglobulins are presented_HAnd V_LAmino acid sequence of the CDR regions of the chain. Table 1 below associates sequence IDs with their respective sequences.

TABLE 1

Brief Description of Drawings

Figure 1. commercially available assays (from Biolegend) for detecting APRIL are not able to reproduce the quantification of APRIL in the presence of serum. Human sera from colorectal cancer patients contained varying amounts of human APRIL, detected by ELISA based on BCMA-Fc coating and polyclonal antibodies for detection (x-axis, as described by planeles et al, haematologic.2007 sep; 92 (9): 1284-5) or detected with a commercial Biolegend ELISA (y-axis). Comparison between the two values for each patient revealed a limited correlation described by the spearman correlation coefficient (R-0,5964).

Figure 2 APRIL quantification using the current commercially available assay is negatively affected by the presence of human serum. Two standard curves were generated using recombinant APRIL at concentrations 100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.136 and 0.04ng/ml diluted with PBS + 10% fetal calf serum + 20% Human Serum (HS), referred to as PBS-FCS-HS, or PBS/1% BSA, referred to as PBS/BSA. The detection of these two standard curves was determined in the following analysis: A) biolegend ELISA, B) coated with BCMA-FC and detected with APRILY-5 biological antibody, C) coated with Sasha-2 and detected with APRILY-5 antibody, and D) R & D ELISA.

Figure 3. selection strategy to identify APRIL-binding peptides that allow detection in serum. Magnetic beads (MagneticDynaBeads) were loaded with BCMA-Fc recombinant protein and after sufficient washing, the BCMA-Fc recombinant protein was allowed to bind to recombinant FLAG-APRIL.

FIG. 4. monoclonal assays for APRIL of the invention in the presence of serum. APRIL binding monoclonal antibodies are used to detect APRIL in CLL patient serum. Sera from three independent patients (CLL1, CLL3, and CLL6) were used, which represent different amounts of APRIL. All APRIL-binding antibodies exhibited similar detection for APRIL in the three different patients, independent of the BCMA-Fc coating to capture APRIL (compare top (500 ng/well BCMA-Fc) to bottom (100 ng/well BCMA-Fc)). B. The antibody hapril.133 was used to determine the detection of APRIL in the serum of CLL patients using more than ten individual samples (CLL11-20) representing different amounts of APRIL.

FIG. 5 quantitation of APRIL using BCMA-Fc and the APRIL binding peptide of the invention (hAPRIL.133mAb) was not affected by the presence of human serum. Two standard curves were generated using recombinant APRIL at concentrations 100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.136 and 0.04ng/ml diluted in PBS + 10% fetal calf serum + 20% Human Serum (HS), referred to as PBS-FCS-HS, or in PBS/1% BSA, referred to as PBS/BSA.

Detailed Description

In the method of the invention for obtaining APRIL-binding peptides, a library of binder peptides is provided. The term "library" is known in the art, and in the known meaning of the term "library of binder peptides" can be understood to mean a collection or array of different binder peptides. The term "conjugate peptide" or alternatively "binding peptide" within the context of a peptide library may be understood as referring to a peptide having the potential ability to bind to other compounds and/or structures, in particular epitopes, more in particular peptide epitopes. In the present invention, in particular, the conjugate peptide has potential APRIL binding ability.

Antibodies (immunoglobulins) and binding fragments thereof are known peptides having the potential to bind other compounds and/or structures, including epitopes, such as peptide epitopes. Thus, in the present invention, in particular, it is envisaged to provide a library of antibodies or fragments thereof. The skilled person knows how to obtain and thus how to provide a library of antibodies or fragments thereof.

For example, antibodies or fragments thereof can be isolated from the generated antibody phage libraries using techniques described in the following references: McCafferty et al, 1990, Nature, 348: 552-554. Clackson et al, 1991, Nature, 352: 624-: 581-597, which respectively describe the isolation of murine and human antibodies using phage libraries. Subsequent publications describe the generation of high affinity (nM range) human antibodies by strand displacement (Marks et al, 1992, Bio/Technology, 10: 779-.

Antibodies or fragments thereof can be isolated from the resulting mRNA display library using techniques described in the following references: fukuda et al, 2006, nuc. acids res, 34: e127, which describes the isolation of antibody fragments using mRNA display libraries.

Alternatively, the antibody library may comprise a collection of lymphocytes, preferably splenocytes, collected from a mammal, such as a non-human mammal, immunized with a substance (agent) suitable for eliciting an APRIL-specific immune response in the mammal. Immunization of (non-human) mammals and collection of spleen cells (or other lymphocytes) are routine practice in the art. The substance suitable for eliciting an APRIL-specific immune response for immunization may be an APRIL protein or a part thereof, in particular in pure form, most preferably in substantially pure form. Alternatively, immunization may be achieved by DNA immunization using a nucleotide sequence encoding APRIL or a portion thereof, preferably a cDNA sequence. Methods and procedures for DNA immunization (procedure) are known to the skilled worker. Exemplary manipulations of DNA immunization are presented in the examples.

In addition to libraries of antibodies (or antibody fragments), libraries of binding peptides engineered on non-immunoglobulin protein backbones can be provided. Examples of such protein scaffolds include, but are not limited to, Adnectins, Affibodies, antibodies, and DARPins (Gebauer and Skerra, Current opinion chem. biol., 2009, 13: 245-. Selection methods include, for example, phage display to identify protein backbones that express APRIL-binding peptides.

In addition, combinatorial peptide libraries can be provided as a library of binder peptides. For example, a combinatorial library of one bead-one compound is a library of peptides that express a large set of peptides on a bead, where one bead binds one peptide. After the selection procedure, the beads are recovered and the peptides are identified using, for example, mass spectrometry (Lam et al, Methods, 1996, 9: 482-93; Xiao et al, comb. chem. high-stem output Screen, 2013, Mar 13(epub ahead of print).

In the method for obtaining APRIL-binding peptides, peptides that specifically bind APRIL are selected from a library of conjugate peptides by means of affinity selection. Affinity selection procedures utilize target peptides immobilized on a solid support. When referring to ligand/receptor, antibody/antigen or other binding pairs, "specifically" binding indicates a binding process that determines the presence of a protein (e.g., APRIL) in a heterologous population of proteins and/or other biological preparations. Thus, under the conditions specified, the specific ligand/antigen binds to a particular receptor/antibody and does not significantly bind to other proteins present in the sample.

The target peptide comprises a plurality of APRIL epitopes and APRIL receptor binding regions of APRIL. The APRIL epitope is preferably derived from a region outside the APRIL receptor binding region of APRIL. Clearly, a number of APRIL epitopes and APRIL receptor binding regions of APRIL may suitably be provided in the form of native APRIL or a derivative thereof. Preferably, natural April is used as the target peptide.

Affinity selection procedures using immobilized ligands for selecting the binder peptide are known in the art. Such as panning or biopanning operations are known. It is known and clear to the skilled person that the usual affinity selection operation comprises three steps: capture, washing and identification of captured binding agents.

For affinity selection procedures employed in the methods of the invention, the capture step involves binding of the library's binder peptides to a target peptide comprising the APRIL receptor binding region of APRIL. It is known in the art that APRIL (currently) has two known natural receptors, BCMA and TACI. Thus, the term APRIL receptor is understood to mean BCMA or TACI. Although the present invention is largely exemplified using BCMA as the APRIL receptor, the use of TACI is equally suitable. The APRIL region of the natural receptor involved in binding APRIL is known (Hymowitz et al, 2005, J.biol.Chem280: 7218-7227). The target peptide comprises the APRIL receptor binding region of APRIL in a form that allows binding to the APRIL receptor (or a binding equivalent thereof). The target peptide is immobilized on a solid support to allow identification and/or isolation of a binder peptide that specifically interacts with the selected target. The term "fixed" is understood to mean having limited or reduced fluidity. The limited or reduced flowability is related to the wash medium used in the washing step. An "immobilized" target peptide need not be directly bound to or interact with a solid support. Rather, it may have an interaction with a compound or moiety that is bound to or interacts with a solid support. Examples of peptide immobilization means include, but are not limited to, non-specific adsorption to plastic, NH 2-coupling to beads, binding to tosyl activated beads, or binding to protein A beads. Methods for immobilizing peptides on solid supports will be clear to the skilled person.

The target peptide in the affinity selection procedure employed in the methods of the invention comprises the APRIL receptor binding region of APRIL and a number of APRIL epitopes. The APRIL receptor binding region of APRIL may be present as the entire APRIL protein or as part of the APRIL protein. The sequence of the APRIL protein or a portion thereof is preferably of human origin. The APRIL epitope may be present in the APRIL receptor binding region of APRIL or in a different portion of the target peptide. The APRIL receptor binding region of APRIL and the choice of APRIL epitopes make possible the binding interaction of the target peptide with the shielding peptide. In this specification and in the claims which follow, unless expressly stated otherwise, each time the term "a number" is used it is to be understood as meaning one or more, such as 1, 2,3, 4, 5, 6,7 or more.

In the method of the invention for obtaining an APRIL-binding peptide, the peptide of interest (comprising the APRIL-binding region of APRIL and a number of APRIL epitopes) is immobilized on a solid support in such a way that it interacts with a shielding peptide comprising the APRIL-binding region of the APRIL receptor or an APRIL-binding equivalent thereof. The APRIL-binding domain of the APRIL receptor may be present as the complete APRIL receptor protein or as part of the APRIL receptor protein. The sequence of the APRIL receptor protein or a portion thereof is preferably of human origin.

As an alternative to APRIL-binding regions for APRIL receptor proteins, APRIL-binding regions of APRIL-binding equivalents of such APRIL receptor proteins may be used. APRIL-binding equivalents of the APRIL receptor protein can be, for example, peptides, such as antibodies, that bind to APRIL at the APRIL-APRIL receptor interface. Such peptides may be selected from peptides that interfere with the interaction of APRIL and many of its receptors (BCMA or TACI). For example, the hapril.01a antibody or analog thereof disclosed in WO 2010/100056. Analogs of hapril.01a are antibody analogs, specifically antibody fragments, as defined in the specification. Whether certain APRIL-binding peptides (such as APRIL-binding antibodies) interfere with the interaction of APRIL and the APRIL receptor can be determined according to the methodology described in example 2 of WO2010/100056 for "receptor blockade".

It should be noted that the target peptide may be immobilized on the solid support by its interaction with the shielding peptide, which is immobilized on the solid support by known means exemplified above.

The washing step follows the capture step. In this step, the unbound components (conjugate peptide and/or target peptide and/or shielding peptide and/or any other components) are washed from the solid support by using a washing medium (e.g., a washing solution). By selecting the washing conditions, the stringency of the selection can be ensured. Such methods will be clear to the skilled person. For example, washing operations involving cells use phosphate buffered saline or culture medium as the washing solution. The wash solution may contain high salt (e.g., 1M sodium chloride) or low salt (e.g., 50mM sodium chloride) to affect the stringency (ionic strength) of the washing operation. The wash solution may also contain a detergent (e.g., Nonidet detergent P-40) to affect the stringency of the washing operation (hydrophobic strength).

In the identification step after the washing step, the binder peptide that still interacts with the target peptide after the washing step is identified. The step of identifying may comprise: the conjugate peptide is eluted from the solid support, after which the eluted conjugate peptide may be identified in any suitable manner known. The skilled artisan can apply mass spectrometry to identify peptides, sequence RNA to identify RNA molecules encoding the binder peptide or sequence DNA to identify cDNA molecules encoding the binder peptide. Alternatively, identification can be performed by using a labeling moiety (e.g., a fluorescent label) attached to the conjugate peptide or the target peptide, such as in a biological microarray application.

According to certain embodiments, the methods of the invention may further comprise a step of negative selection of peptides bound to the solid support and/or the shielding peptide. In certain affinity selection procedures, the use of such negative selection steps may result in APRIL-binding peptides with improved specificity for APRIL. The improvement is associated with APRIL-binding peptides obtained in a process that does not include a negative selection step.

In the negative selection step, the conjugate peptide is discarded if its affinity for the shielding peptide or the solid support is higher than its affinity for the target peptide. The negative selection step can be performed using the target peptide interacting with the shielding peptide before or after the capture step (initial capture step). According to certain embodiments, the negative selection step is performed by a negative capture step comprising binding of the binder peptides of the library to the shielding peptide (immobilized on a solid support) in the absence of the target peptide prior to the initial capture step. In this negative preselection step, unconjugated conjugate peptides are selected for the initial step. According to certain other embodiments, after the initial capture step, the negative selection step is performed by a negative capture step comprising binding of the conjugate peptide selected in the initial capture step in the absence of the target peptide to the shielding peptide (immobilized on a solid support). In this negative post-selection step, unconjugated conjugate peptide is selected as APRIL-binding peptide. For the negative selection step, it is preferred that the immobilization of the target peptide is dependent on its interaction with the shielding peptide (the shielding peptide interacts more strongly with the solid support than with the target peptide). In this embodiment, after the pre-selection assay, the target peptide may be brought into interaction with the immobilized shielding peptide on the solid support, or the interaction of the target peptide and the immobilized shielding peptide may be perturbed for a post-selection step.

In the practice of the methods of the invention described above, APRIL-binding peptides are identified and/or isolated. To facilitate the production of APRIL-binding peptides, it is advantageous to determine the amino acid sequence of the selected APRIL-binding peptide and/or the nucleotide sequence encoding the amino acid sequence of an APRIL-binding peptide identified and/or obtained by the present method. This enables the transfection of nucleotide sequences encoding APRIL-binding peptides to produce organisms capable of producing APRIL-binding peptides with good efficacy. Depending on the library of binder peptides used, the skilled person may use a variety of methods to determine and/or isolate the nucleotide sequence encoding the APRIL-binding peptide.

In the case where the library is a collection of lymphocytes collected from an immunized mammal, the APRIL-binding peptide is an immunoglobulin molecule presented on the cell surface of the obtained lymphocyte clone. The nucleotide sequence encoding an APRIL-binding peptide can be obtained by: RNA was isolated from cultures of lymphocyte clones, immunoglobulin sequences were selectively amplified using immunoglobulin-specific primers, and the selectively amplified sequences were subsequently sequenced.

Where the library is a collection of phage, the binding peptide selected is an antibody or antibody fragment presented on the surface of the phage. The nucleotides encoding the APRIL-binding peptide may be isolated by: isolating DNA from the isolated phage followed by sequencing the DNA.

In case the library is a collection of ribosomally displayed mrnas, the selected binding peptide is displayed on the ribosome. The nucleotides encoding the APRIL-binding peptide can be isolated by isolating mRNA that binds to ribosomes. The identity (identity) of the binding peptide can be determined by the following steps: the RNA is sequenced directly or cDNA complementary to the mRNA is generated, followed by sequencing of the selected amplified sequence.

In the case where the library is a collection of bead-bound binding peptides (a bead-compound library), one binding peptide is bound to one bead. The properties of APRIL-binding peptides are determined by the following steps: peptides are recovered from beads selected in an affinity selection procedure, followed by a mass spectrometry procedure.

It is clear that in the method of the invention for obtaining APRIL-binding peptides, reactions and processes such as binding affinity selection processes and related processes such as capture steps and wash steps can be performed in a suitable container, such as a reactor, in particular a vessel used in the laboratory scale of such screening methods.

The invention also relates to APRIL-binding peptides obtained by the method of the invention for obtaining APRIL-binding peptides. It will be clear to the skilled person that with the method of the invention a large number of different APRIL-binding peptides can be obtained. The binding peptides obtained with the method of the invention share common features: they have reduced interference with the binding of APRIL to its receptor compared to known APRIL-binding peptides. They are therefore better able to bind APRIL in complex with APRIL receptors or their binding equivalents (e.g., hapril.01a or analogs thereof). The K of an APRIL-binding peptide (e.g., an antibody) of the invention is generally directed against its target (APRIL, preferably human APRIL)_DAbout less than 10^-3M, more typically less than 10^-6M, usually less than 10^-7M, more typically less than 10^-8M, preferably less than 10^{- 9}M, and more preferably less than 10^-10M and most preferably less than 10^-11M (see, e.g., Presta, et al, 2001, Thromb. Haemost.85: 379-. According to certain embodiments, the K of an APRIL-binding peptide (e.g., an antibody) of the invention to its target (APRIL, preferably human APRIL)_DCan be selected from 1 × 10^-6To 0.5X 10^-11M、1×10^-7To 0.5X 10^-11M、1×10^-8To 0.5X 10^-11M、1×10^-8To 1X10^-11M, preferably 5X10^-9To 1X10^-11M, more preferably 5X10^-9To 1X10^-10And M. Binding affinity can be determined using standard assaysAnd a force. According to certain embodiments, the APRIL-binding peptides obtained have an inhibitory IC50 for APRIL receptor-APRIL interaction of at least 5x10^-9M, preferably higher than 1X10^-8M, more preferably higher than 1X10^-7M and most preferably higher than 1X10^-6And M. For example, inhibition of the APRIL receptor-APRIL interaction with IC50 can be 5X10^-9To 1X10^-4M, e.g. 5X10^-9To 1X10^-5M, preferably 1X10^-8To 1X10^-5M, e.g. 1X10^-8To 1X10^-6M，1×10^-8To 1X10^-7M，1×10^-7To 1X10^-5M, or 1X10^-7To 1X10^-6M。

According to certain embodiments, the APRIL-binding peptide obtained is an immunoglobulin or a binding fragment thereof. In the present description and the claims, the terms immunoglobulin and antibody are used as synonyms and are therefore interchangeable. The term "antibody" refers to any form of antibody that exhibits the desired activity, specifically binding to a target. By binding to the target, certain desired effects may be promoted. For example, a related compound or moiety may be targeted to a target location, for example, by binding to an antibody. In the present invention, the target is APRIL, preferably human APRIL. An antibody that targets APRIL can bind APRIL when APRIL binds to its receptor or analog.

The term "antibody" is used in its broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies). In the present invention, peptides derived from certain antibodies may be considered antibody analogs. The skilled artisan understands that for proper function of an antibody analog within the context of the present invention, the resulting antibody (or antibody analog) comprises the antigen binding region of its original antibody. In particular, antibody analogs include antibody fragments, antibodies with improved effector function, chimeric antibodies and humanized antibodies as defined below.

"antibody fragment" and "antibody-binding fragment" mean the antigen-binding fragment and comparable portions of an antibody, typically comprising at least part of the antigen-binding junction of the parent antibodyA fixed or variable domain. Antibody fragments retain at least some of the binding specificity of the parent antibody. For this case, the antibody fragment comprises a plurality of CDRs, in particular V_HMany CDRs of a region, e.g. V_HCDR1, CDR2, and CDR3 of the region. Except for V_HIn addition to many CDRs of a region, an antibody fragment may also comprise V_LMany CDRs of a region, e.g. V_LCDR1, CDR2, and CDR3 of the region. According to certain embodiments, the antibody fragment may comprise a peptide with V_LV binding of CDR1, CDR2 and CDR3 of the region_HCDR1, CDR2, and CDR3 of the region. Typically, an antibody fragment retains at least 10% of the parent binding activity when expressed on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target. Thus, it will be clear to the skilled person that "antibody fragments" may be substituted for antibodies in many applications, and that the term "antibody" should be understood to include "antibody fragments" where such substitution is appropriate. Examples of antibody fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments; a bivalent antibody; a linear antibody; single chain antibody molecules, such as sc-Fv, single antibodies (unibodes) or diabodies (duobodes) (techniques from Genmab); domain antibodies (technology from domentis); nanobodies (technology from Ablynx); and multispecific antibodies formed from antibody fragments. Engineered antibody variants are reviewed in Holliger and Hudson, 2005, nat. biotechnol.23: 1126-1136.

"Fab fragment" consists of one light chain and one heavy chain C_HRegion 1 and variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The "Fc" region contains a C comprising an antibody _H1 and C_H2 domain. Two heavy chain fragments through two or more disulfide bonds and through C_HThe hydrophobic interactions of the 3 domains remain together.

"Fab' fragment" contains one light chain and contains V_HDomains and C _H1 domain and C _H1 and C_H2 domains, so that it can be between the two heavy chains of the two Fab' fragmentsInterchain disulfide bond to form F (ab')₂A molecule.

“F(ab')₂Fragment "contains two light chains and two contains C _H1 and C_H2 domain such that an interchain disulfide bond is formed between the two heavy chains. Thus, F (ab')₂The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

The "Fv region" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

"Single chain Fv antibody" (or "scFv antibody") refers to a V comprising an antibody_HAnd V_LAntibody fragments of domains, wherein the domains are present on a single polypeptide chain. In general, the Fv polypeptide further comprises V_HAnd V_LA polypeptide linker between the domains which enables the scFv to form the desired structure for antigen binding. For an overview of scFv see Pluckthun, 1994, the Pharmacology of monoclonal Antibodies, Vol 113, Rosenburg and Moore, Springer-Verlag, New York, page 269-315. See also international patent application publication No. WO 88/01649 and U.S. Pat. nos. 4,946,778 and 5,260,203.

A "diabody" is a small antibody fragment with two antigen-binding sites. Said fragments comprising the same polypeptide chain (V)_H-V_LOr V_L-V_H) Light chain variable domain of (V)_L) Linked heavy chain variable domains (V)_H). By using a linker that is too short to allow pairing of the two domains on the same strand, the domains are forced to pair with the complementary domains of the other strand and two antigen binding sites are created. Bivalent antibodies are more fully described in, for example, EP 404,097; WO 93/11161; and Holl iger et al, 1993, proc.natl.acad.sci.usa 90: 6444-6448.

A "domain antibody fragment" is an immunologically functional immunoglobulin fragment that contains only heavy chain variable regions or light chain variable regions. In certain examples, two or more V_HThe regions are covalently linked to a peptide linker to produce a bivalent domain antibody fragment. Bivalent domain antibodyTwo V of a fragment_HThe regions may target the same or different antigens.

The antibody fragments of the invention may comprise a constant region portion sufficient to allow dimerization (or multimerization) of heavy chains having reduced disulfide bonding capability, for example wherein at least one of the hinge cysteines normally involved in the inter-heavy chain disulfide bond is altered by known methods available to the skilled person. In another embodiment, an antibody fragment (e.g., one comprising an Fc region) retains at least one biological function normally associated with an Fc region present in an intact antibody, such as binding of FcRn, half-life modulation of the antibody, ADCC (antibody dependent cellular cytotoxicity) function, and/or complement binding (e.g., wherein the antibody has glycosylation properties necessary for ADCC function or complement binding).

In the present invention, the antibody is directed against APRIL, preferably human APRIL, and thus comprises a binding domain that binds to and/or interacts with APRIL, preferably human APRIL. The antibody may be raised in a mammal of a non-human species suitable for raising antibodies against human antigens. Alternatively, the antibodies can be isolated from the generated antibody phage libraries using techniques described in the following references: McCafferty et al, 1990, Nature, 348: 552 and 554; clackson et al, 1991, Nature, 352: 624-: 581-597. The skilled person is able to select a suitable non-human species for eliciting antibodies against human antigens. For example, the selection may be made in a non-human mammal, such as a rodent, including murine (rat or mouse) or hamster species, or alternatively a camelid species.

The antibodies, when raised in non-human species, are preferably chimerized to form "chimeric antibodies" using methods and techniques known in the art.

The term "chimeric" antibody refers to an antibody that: wherein part of the heavy and/or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No.4,816,567 and Morrison et al, 1984, Proc. Natl. Acad. Sci. USA 81: 6851-6855). In the present invention, the "chimeric antibody" is preferably a "humanized antibody".

As used herein, the term "humanized antibody" refers to a form of an antibody that contains sequences from non-human (e.g., murine) as well as human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. In general, a humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally further comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Humanized forms of rodent antibodies comprise essentially the same CDR sequences of the parent rodent antibody, although substitutions of certain amino acids may be included to increase affinity, increase stability of the humanized antibody or for other reasons. However, because the exchange of CDR loops results unevenly in antibodies with the same binding properties as the antibody of origin, changes in Framework Residues (FR), residues involved in the support of CDR loops can also be introduced into humanized antibodies to maintain the binding affinity of the antigen (Kabat et al, 1991, J.Immunol.147: 1709).

The term "antibody" also includes "fully human" antibodies, i.e., antibodies that comprise only the protein sequences of a human immunoglobulin. A fully human antibody may contain sugar chains of non-human, such as murine (rat or mouse), if it is produced in a mouse, non-human cell (e.g., mouse or hamster), or in a hybridoma derived from a murine cell. Likewise, "mouse antibody" or "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans, in transgenic non-human animals with human immunoglobulin germline sequences, by phage display or other molecular biological methods. Likewise, recombinant immunoglobulins can also be produced in transgenic mice. See Mendez et al, 1997, Nature Genetics 15: 146-156. See also the techniques of Abgenix, Medarex, MeMo and Kymab.

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs" defined by sequence alignment, for example, residues 24-34(L1), 50-56(L2) and 89-97(L3) of the light chain variable domain and residues 31-35(H1), 50-65(H2) and 95-102(H3) (see Kabat et al, 1991, Sequences of proteins of immunological interest, fifth edition, Public Health Service, National Institutes of Health, Besda, Md.) and/or residues structurally defining the "hypervariable loop" (HVL), for example, residues 26-32(L1), 50-52(L2) and 91-96(L3) of the light chain variable domain and residues 26-32(H1), residues 53-55(H2) and residues 96 (H4655-2) of the heavy chain variable domain (H49325 and H3 (H3), 1987, J.mol.biol.196: 901-917). "framework" or "FR" residues are variable domain residues other than the hypervariable region residues defined herein.

According to certain embodiments, the obtained APRIL-binding peptide (e.g., an antibody or analog thereof) comprises an immunoglobulin V_HDomain of an immunoglobulin V_HThe domain comprises a sequence identical to a sequence selected from SEQ ID NO: 5. 6 and 7, or SEQ ID NO: 15. 16 and 17, or SEQ ID NO: 25. 26 and 27, or SEQ ID NOs: 35. 36 and 37, or SEQ ID NOs: 45. 46 and 47 have a CDR1, CDR2 and CDR3 sequence having at least 60%, such as at least 85%, preferably at least 90%, more preferably at least 95% sequence similarity, such as V_HThe domain has at least 60%, such as at least 85%, preferably at least 90%, more preferably at least 95% sequence similarity to an amino acid sequence selected from the group consisting of SEQ ID No.3, 13, 23, 33 or 43. The skilled person understands that V_HThe domains primarily govern the binding affinity and specificity of the antibody. Thus, as in camelid-derived and camelized antibodies, in V_LEfficient binding can be obtained in the absence of a domain.

The APRIL-binding peptide (e.g., an anti-APRIL antibody or analog thereof) may comprise an immunoglobulin V_HAnd V_LDomain ofThe immunoglobulin V_HAnd V_LThe domain comprises a sequence identical to a sequence selected from SEQ ID NO: 5. 6,7, 8, 9 and 10, or SEQ ID NO: 15. 16, 17, 18, 19 and 20, or SEQ ID NO: 25. 26, 27, 28, 29 and 30, or SEQ ID NO: 35. 36, 37, 38, 39 and 40, or SEQ ID NO: 45. 46, 47, 48, 49 and 50 has a V of at least 60%, such as at least 85%, preferably at least 90%, more preferably at least 95% sequence similarity_HCDR1、V_HCDR2、V_HCDR3、V_LCDR1、V_LCDR2 and V_LCDR3 sequences, e.g. V_HAnd V_LA domain pair to a sequence selected from SEQ ID NO: 3 and 4, or 13 and 14, or 23 and 24, or 33 and 34, or 43 and 44, have at least 60%, such as at least 85%, preferably at least 90%, more preferably at least 95% sequence similarity. The coding DNA sequences of these various sequences can be determined by the skilled worker on the basis of the knowledge of the genetic code. In Table 2 below, V is shown_HAnd V_LMany coding DNA sequences of amino acid sequences. The sequences are provided in the sequence listing.

The skilled artisan understands that "sequence similarity" refers to the degree to which the individual nucleotide or peptide sequences are identical. The degree of similarity between two sequences is based on the degree of identity combined with the degree of conservative variation. The percentage of "sequence similarity" is the percentage of amino acids or nucleotides that are identical or conservatively varied, i.e., "sequence similarity" (% sequence identity) + (% conservative variation).

For the purposes of the present invention, "conservative variations" and "identity" are considered to be a broader category of the term "similarity". Thus, whenever the term "sequence similarity" is used, it encompasses sequence "identity" and "conservative changes".

The term "sequence identity" is known to the skilled person. To determine the degree of sequence identity shared by two amino acid sequences or by two nucleic acid sequences, the sequences are aligned for optimal alignment purposes (e.g., gaps are introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). Such alignment can be performed over the full length sequences being compared. Optionally, the alignment is performed on nucleic acids/bases or amino acids of shorter comparative lengths, e.g., about 20, about 50, about 100, or more.

The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are equivalent at that position. The degree of identity shared between sequences is typically expressed in terms of percent identity between the two sequences and is a function of the number of equivalent positions shared by the equivalent residues of the sequences (i.e.,% identity ═ the number of equivalent residues at the corresponding positions/total number of positions x 100). Preferably, the two sequences being compared are two sequences of the same or substantially the same length.

The percentage of "conservative changes" can be determined in a manner similar to the percentage of sequence identity. However, in this example, changes at specific positions of the amino acid or nucleotide sequence that are likely to retain the functional properties of the starting residue are scored as if no change had occurred.

For amino acid sequences, the relevant functional property is the physicochemical property of the amino acid. Conservative substitutions of amino acids in a polypeptide of the invention may be selected from other members of the class to which the amino acid belongs. For example, protein biochemistry is well known in the art, where amino acids belonging to a group of amino acids having a particular size or characteristic (e.g., charge, hydrophobicity, and hydrophilicity) can be replaced by another amino acid without substantially altering the activity of the protein, particularly in regions of the protein not directly related to biological activity (see, e.g., Watson, et al, Molecular Biology of the Gene, the Benjamin/Cummings pub. Co., p.224 (fourth edition 1987)). For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negative charge(acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, substitution of Lys to Arg and vice versa to maintain a positive charge; replacement of Glu with Asp and vice versa to maintain negative charge; replacement of Ser to Thr and vice versa allows the free-OH to be maintained; and replacement of Gln by Asn and vice versa to maintain free-NH₂。

Exemplary conservative substitutions in the amino acid sequence of an APRIL-binding peptide of the present invention may be made as follows:

exemplary conservative amino acid substitutions

For nucleotide sequences, the relevant functional properties are primarily biological information: certain nucleotides are carried in the open reading frame of sequences involved in the transcriptional and/or translational machinery. It is common knowledge that genetic codons are degenerate (or redundant) and that multiple codons can carry the same information relating to the amino acid they encode. For example, in certain species, the amino acid leucine may be encoded by UUA, UUG, CUU, CUC, CUA, CUG codon (or TTA, TTG, CTT, CTC, CTA, CTG for DNA), and the amino acid serine is specified by UCA, UCG, UCC, UCU, AGU, AGC (or TCA, TCG, TCC, TCT, AGT, AGC for DNA). Nucleotide changes that do not alter the translational information are considered conservative changes.

The skilled person is aware of the fact that: several different computer programs (using different mathematical algorithms) can be used to determine the identity between two sequences. For example, the use may consist of a computer program that employs the Needleman and Wunsch algorithm (Needleman et al (1970)). According to one embodiment, the computer program is the GAP program in the Accelerys GCG software package (Accelerys inc., San Diego u.s.a). Alternative matrices that may be used are, for example, the BLOSUM62 matrix or the PAM250 matrix, the null weights 16, 14, 12, 10, 8, 6, or 4, and the length weights 1, 2,3, 4, 5, or 6. The skilled person understands that all these different parameters will yield slightly different results when using different algorithms, but that the total percent identity of the two sequences is not significantly changed.

According to one embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the Accelrys GCG software package (accelerloreys inc., San diegou.s.a). The NWSgapdna CMP matrix is used with gap weights 40, 50, 60, 70 or 80 and length weights 1, 2,3, 4, 5 or 6.

In another embodiment, the percentage identity of two amino acid or nucleotide sequences is determined using the PAM120 weight residue table, gap length penalty 12 and gap penalty 4 using the algorithms of E.Meyers and W.Miller (Meyers et al (1989)) which have been incorporated into the ALIGN program (version 2.0) (available at the ALIGN Query using sequence data of the Gene streamserver IGH Montpell ier France (http:// vegajgh. mrs. fr/bin ALIGN-gain. cgi)).

For the present invention, it is most preferred to use BLAST (basic Local Alignment tool) to determine percent identity and/or similarity between nucleotide or amino acid sequences. Queries using the BLASTn, BLASTp, BLASTx, tBLASTnt and BLASTx programs of Altschul et al (1990) can be published via http:// www.ncbi.nlm.nih.gov on-line version BLAST. Alternatively, a standalone version of BLAST { e.g., version 2.2.24 (published on 23/8/2010) may be used, also downloaded via the NCBI website). Preferably, the BLAST query is performed with the following parameters. To determine percent identity and/or similarity between amino acid sequences: the algorithm is as follows: blastp; word length: 3; a scoring matrix: BLOSUM 62; vacancy cost: there are: 11, extension: 1; composition adjustment: adjusting a condition composition scoring matrix; a filter: closing; mask (mask): and closing. To determine percent identity and/or similarity between nucleotide sequences: the algorithm is as follows: blastn; word length: 11; maximum match of query range: 0; match/mismatch score: 2, -3; vacancy cost: there are: 5, extension: 2; a filter: a low complexity region; masking: only the look-up table is masked.

The percentage of "conservative changes" can be determined by means of the algorithm and computer program shown, in a similar percentage to the sequence identity. Certain computer programs, e.g., BLASTp, represent the number/percentage of positives (═ similarities) and the number/percentage of identities. The percentage of conservative changes can be obtained by: the percentage of identity is subtracted from the percentage of positive/similarity (percent conservative change-percent identity).

Further manipulations are possible based on the available sequence information for APRIL-binding peptides. If the APRIL-binding peptide is an antibody, the antibody DNA may also be modified, for example, by replacing the coding sequences with the constant regions of the human heavy and light chains in place of the homologous murine sequences (U.S. Pat. No.4,816,567; Morrison, et al, 1984, proc. natl acad. sci. usa, 81: 6851), or by covalently linking the immunoglobulin coding sequence to all or part of the coding sequence of a non-immunoglobulin substance (e.g., a protein domain). Typically such non-immunoglobulin substances are replaced with constant domains of an antibody, or with variable domains of one antigen-binding site of an antibody to produce a chimeric bivalent antibody comprising one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

Camelized antibodies are heavy chain-only antibodies derived from mouse antibodies. The expression may be expressed as Tanha et al, Protein EngDes sel, 2006, 19: 503-9 for camelization.

Humanized antibodies have one or more amino acid residues from a non-human source. Non-human amino acid residues are often referred to as "import" residues, and they are typically taken from an "import" variable domain. Humanization is generally performed according to the method of Winter and co-workers (Jones et al, 1986, Nature 321: 522-525; Riechmann et al, 1988, Nature, 332: 323-327; Verhoeyen et al, 1988, Science 239: 1534-1536) by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are antibodies that: wherein substantially less than one entire human variable domain is substituted with the corresponding sequence from the non-human species. In practice, humanized antibodies are typically human antibodies in which certain CDR residues and possibly certain FR residues are substituted by residues from analogous positions in non-human, e.g., rodent, antibodies.

The choice of human variable domains (hydrogen and heavy chains) used to make the humanized antibody is important if reduced antigenicity is relevant. The sequence of the variable domain of a rodent antibody is screened against a full-text library of known human variable-domain sequences according to the so-called "best-match" method. The human sequence closest to the rodent sequence was then used as the human Framework (FR) for the humanized antibody (Sims et al, 1987, J.Immunol.151: 2296; Chothia et al, 1987, J.mol.biol.196: 901). Another approach uses a specific framework derived from the common sequence of all human antibodies of a specific subclass of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al, 1992, Proc. Natl. Acad. Sci. USA 89: 4285; Presta et al, 1993, J. Immunol.151: 2623).

It is further important that antibodies be humanized so that they retain high affinity for the antigen as well as other beneficial biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analyzing the parent sequence and various conceptual humanized products using three-dimensional models of the parent sequence and the humanized sequence. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available which exemplify and show the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Viewing these displays allows analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences such that the desired antibody characteristics, such as increased affinity for the target antigen, are achieved. In general, CDR residues are directly and maximally involved in affecting antigen binding.

Humanization of antibodies is a straightforward protein engineering task. Almost all murine antibodies can be humanized by CDR grafting, resulting in the retention of antigen binding (see Lo, Benny, K.C., editor, in antibody engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004).

Amino acid sequence variants of the humanized anti-APRIL antibody are prepared by introducing appropriate nucleotide changes into the DNA of the humanized anti-APRIL antibody or by peptide synthesis. Such variants include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence shown in the humanized anti-APRIL antibody. Any combination of deletions, insertions, and substitutions are made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes may also alter post-translational processes of the humanized anti-APRIL antibody, such as changing the number or position of glycosylation sites.

A useful method for identifying certain residues or regions of a humanized anti-APRIL antibody polypeptide, which are preferred positions for mutagenesis, is referred to as "alanine scanning mutagenesis", e.g., Cunningham and Wells, 1989, Science 244: 1081-1085. Wherein a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) of the target residue is identified and substituted with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the APRIL antigen. Amino acid residues that exhibit functional sensitivity to the substitution are then improved by introducing additional or other mutations at the position of the substitution. Thus, when the site for introducing an amino acid sequence change is predetermined, the nature of the mutant itself need not be predetermined. For example, to analyze the performance of mutations at a given site, alanine scanning or random mutagenesis is performed at the target codon or target region and the expressed humanized anti-APRIL antibody is screened for variants according to the desired activity.

Typically, the amino acid sequence mutation of the humanized anti-APRIL antibody comprises an amino acid sequence that has at least 75% amino acid sequence identity to the amino acid sequence of the heavy or light chain of the original mouse antibody, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably at least 95%, 98% or 99%. Identity or homology with respect to this sequence is defined herein as: after aligning the sequences and introducing gaps, the percentage of amino acid residues in the candidate sequence that are equivalent to the humanized residues, if necessary, to achieve the maximum percent sequence identity, is not considered to be any conservative substitutions as part of the sequence identity. Deletion or insertion of antibody sequences at the N-terminus, C-terminus or internal extension should be construed as not affecting sequence identity or homology. The percent identity between two sequences can be determined in a computer application such as SeqMan II (DNAstar Inc, version 5.05). The two sequences using this program can be aligned using the optimal alignment algorithm of Smith and Waterman (1981) (Journal of molecular Biology 147: 195-197). Percent identity can be calculated by dividing the number of equivalent amino acids between the two sequences by the length of the aligned sequences minus the length of all gaps after the two sequences are aligned.

Antibodies having the characteristics identified herein, which are required for humanized anti-APRIL antibodies, can be screened for in vitro inhibition of biological activity or for suitable binding affinity. To screen for Antibodies on epitopes that bind to human APRIL, a conventional cross-blocking (cross-blocking) assay as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlowand David Lane (1988) can be performed. In such an assay, antibodies that bind the same epitope are likely to cross-block, but not all cross-blocking antibodies necessarily bind at the exact same epitope, as cross-blocking may result from steric hindrance of antibody binding by antibodies binding at overlapping epitopes or even near non-overlapping epitopes.

Alternatively, a procedure such as Champe et al, 1995, j.biol.chem.270: 1388-1394 to determine whether the antibody binds to the target epitope. Also available are the methods described by Cunningham and Wells, 1989, Science 244: "alanine scanning mutagenesis" as described in 1081-1085, some other form of point mutagenesis of amino acid residues in human APRIL, to determine functional epitopes of the anti-APRIL antibodies of the invention. Another method to map the epitopes of antibodies is to study the binding of antibodies to synthetic linear peptides and CLIPS peptides, which can be screened using the credit card model mini PEPSCAN card described in the following documents: medmem et al (WO/2010/100056), Slootstra et al (Slootstra et al, 1996, mol. division 1: 87-96) and Timmerman et al (Timmerman et al, 2007, J.mol. Recognit.20: 283-. The binding of the antibody to each peptide was determined by PEPSCAN-based enzyme-linked immunosorbent assay (ELISA).

For example, additional antibodies that bind the same epitope as an antibody of the invention can be obtained by screening for antibodies that are increased against APRIL based on binding to the epitope, or by immunizing an animal with a peptide comprising a human APRIL fragment containing the epitope sequence. Antibodies that bind to the same functional epitope are expected to exhibit similar biological activities, such as blocking receptor binding, and such activities can be confirmed by functional analysis of the antibodies.

For example, additional APRIL-binding peptides directed to the same epitope as an antibody of the invention can be obtained by preselecting the binding peptides using the selection techniques of the invention and libraries displaying the binding peptides. Binding peptides that bind to the same functional epitope are expected to exhibit similar biological activities, such as blocking receptor binding, and such activities can be confirmed by functional analysis of the antibody.

The affinity of APRIL-binding peptides for APRIL was determined using standard assays. Preferred binding peptides such as antibodies are those with the following K_dAntibodies that bind human APRIL: less than about 1x10^-7M; preferably less than about 1x10^-8M; more preferably less than about 1x10^-9M; and most preferably less than about 1x10^-10M or even less than 1x10^-11M。

As used herein, the term "about" refers to a value within an acceptable error range for the particular value determined by one of skill in the art, which depends in part on how the value is measured or determined, i.e., the limits of the measurement system. For example, "about" can mean within 1 or over 1 standard deviation per practice in the art. Alternatively, "about" or "substantially comprising" may mean a range of up to 20%. Furthermore, particularly in relation to biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the value. Unless otherwise specified, when a particular value is referred to in this application and claims, the meaning of "about" or "consisting essentially of" should be assumed to be within an acceptable error range for the particular value.

The antibody may be selected from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE. Preferably, the antibody is an IgG antibody. Any isotype of IgG may be used, including IgG1, IgG2, IgG3, and IgG 4. Variants of the IgG isotype are also contemplated. Humanized antibodies may comprise sequences from more than one species or isotype. Optimization of the necessary constant domain sequences to produce the desired biological activity is readily accomplished by screening antibodies using the biological assays described in the examples.

Likewise, any species of light chain may be used in the compositions and methods herein. In particular, κ, λ, or variants thereof are used in the compositions and methods of the invention.

The APRIL-binding peptides of the invention (e.g., anti-APRIL antibodies or antibody analogs thereof) may be conjugated to a label, e.g., a label selected from fluorescent or luminescent labels including fluorophores (e.g., rare earth chelates), fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, phycoerythrins, phycocyanins, allophycocyanins, o-phthalaldehyde, fluorescamines, and mixtures thereof,¹⁵²Eu, dansyl, a chromone, fluorescein, a luminal label (luminal label), an isoluminol label (isoluminol label), an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a aequorin label, 2, 3-dihydrophthalazinedione, biotin/avidin, a rotational label, and a stable free radical.

Any suitable method known in the art for conjugating a protein molecule to multiple moieties may be employed, including methods described in the following references: hunter et al, 1962, Nature 144: 945; david et al, 1974, Biochemistry 13: 1014; pain et al, 1981, j. immunol. meth.40: 219; and Nygren, j., 1982, histochem.andcechem.30: 407. methods for conjugating antibodies and proteins are conventional and well known in the art.

According to certain embodiments, the APRIL-binding peptide obtained with the method of the invention is a binding peptide obtained from a combinatorial peptide library. Such APRIL-binding peptides need not be based on antibody structure and thus may be non-antibody-binding peptides. Examples include APRIL-binding peptides derived from a one-bead one-peptide library. Other examples include APRIL-binding peptides based on engineered protein backbones (e.g., Adnectins, Affibodies, anticancer ins, and DARPins).

Other aspects of the invention relate to cells comprising a nucleotide sequence encoding an APRIL-binding peptide obtained by a method of the invention for obtaining an APRIL-binding peptide. As discussed above, the nucleotide sequence encoding the APRIL-binding peptide may be determined and/or isolated by different procedures, depending on the library of binder peptides used. Thus, the nucleotide sequence encoding the APRIL-binding peptide of the invention can be obtained. Such nucleotide sequences may be used for transfection of host cells. Thus, the cell may be a genetically engineered cell. In particular, the cell may be genetically engineered by comprising as heterologous nucleotide sequence a nucleotide encoding an APRIL-binding peptide.

The host cell may be a cloning host or an expression host. When selected as an expression host, the host cell expression system is preferably capable of, and more preferably optimized for, the production of heterologous peptides, such as antibodies or antibody fragments. The host cell may be from a unicellular organism or from a multicellular organism, and may be selected from an escherichia coli cell, a simian COS cell, a Chinese Hamster Ovary (CHO) cell, or a myeloma cell that does not otherwise produce immunoglobulin or APRIL-binding peptides. For transfection, the isolated DNA may be inserted into an expression vector, which is then transfected into a host cell.

Alternatively, it is also possible to generate transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin upon immunization. For example, it has been described that deletion of homozygotes of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrangement in such germline mutant mice will result in the production of human antibodies based on antigenic challenges (see, e.g., Jakobovits et al, 1993, Proc. Natl. Acad. Sci. USA 90: 2551; Jakobovits et al, 1993, Nature 362: 255-258; Bruggemann et al, 1993, Yeast in Immunology 7: 33; and Duchosal et al, 1992, Nature 355: 258).

Using the cells of the invention, APRIL-binding peptides can be produced. Thus, a further aspect of the invention relates to a process for producing an APRIL-binding peptide, comprising providing a cell of the invention, culturing said cell and allowing said cell to express and preferably secrete an APRIL-binding peptide.

APRIL-binding peptides can be isolated from host cell expression systems and various procedures for this process are readily available to the skilled artisan. The particular procedure most suitable will depend on the host cell expression system used and the skilled person will be able to make appropriate selections based on the available common general knowledge.

When using recombinant techniques, APRIL-binding peptides, such as antibodies (or the like), can be produced intracellularly in the periplasmic space or secreted directly into the culture medium. If the APRIL-binding peptide is produced intracellularly, as a first step, particulate debris (host cell or cytolytic fragments) are removed, for example, by centrifugation or ultrafiltration. Carter et al, 1992, Bio/Technology 10: 163-167 describes the procedure for isolating the antibody secreted into the periplasmic space of E.coli. Briefly, the Cell paste (Cell paste) was thawed in the presence of sodium acetate (ph3.5), EDTA and phenylmethylsulfonyl chloride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. When APRIL-binding peptides are secreted into the culture medium, the supernatant from such expression systems is typically first concentrated using commercially available protein concentration filters, e.g., Amicon or Millipore Pellicon ultrafiltration devices. Protease inhibitors such as PMSF may be included in the above steps to inhibit proteolysis, and antibiotics may be added to prevent the growth of adventitious contaminants.

APRIL-binding peptide compositions prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, which are particularly useful purification techniques. The suitability of protein a as an affinity ligand for an immunoglobulin depends on the species and isotype of any immunoglobulin Fc region present in its protein sequence. Protein A can be used to purify antibodies based on the heavy chains of human Ig. λ 1, Ig. λ 2, or Ig. λ 4 (Lindmark et al, 1983, J.Immunol. meth.62: 1-13). Protein G is recommended for all mouse isoformsAnd Ig. λ 3(Guss et al, 1986, EMBO J5: 1567-1575). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also useful. A physically stable matrix such as controlled pore glass or poly (styrene divinyl) benzene allows for faster flow rates and shorter process times than can be achieved with agarose. When the APRIL-binding peptide is an antibody and comprises C _H3 domain, Bakerbond ABX^TMResins (j.t.baker, phillips burg, n.j.) are useful for purification. Other protein purification techniques such as ion exchange column based fractionation, ethanol precipitation, reverse phase HPLC, silica based chromatography, heparin based Sepharose^TMChromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also useful, depending on the antibody being recovered.

APRIL-binding peptides (e.g., immunoglobulins, including binding fragments thereof) obtained by the process of producing APRIL-binding peptides are further aspects of the invention. The APRIL-binding peptides typically have a peptide sequence within the definition of an APRIL-binding peptide obtained by the method of obtaining the APRIL-binding peptide. However, differences may exist in post-translational modifications, such as glycosylation properties. For example, antibodies lacking core fucose residues have been shown to exhibit enhanced ADCC activity. Modulation of glycosylation of antibody patterns is known to the skilled artisan. For example, GlycoFi technology allows for specific tailoring of antibody glycosylation to show the desired level of Fc effector function (Beck et al, Expert Opin Drug discov., 2010, 5: 95-111).

The APRIL-binding peptide obtained by the process of producing an APRIL-binding peptide may be an isolated antibody. An "isolated" antibody is an antibody that is identified and isolated and/or recovered from a component of its natural environment. Impurity components of their natural environment are substances that interfere with diagnostic or therapeutic uses of the antibodies, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the peptide is purified (1) to at least 50%, such as at least 60%, preferably at least 80%, such as at least 90%, for example as determined by the Lowry method, and most preferably more than 95%, such as at least 99%, by weight of the protein in the composition comprising the peptide, (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a rotating cup sequencer, or (3) to exhibit homogeneity by SDS-PAGE under reducing or non-reducing conditions using coomassie brilliant blue or preferably silver staining. Isolating the antibody includes in situ antibody within the recombinant cell, as at least one component of the natural environment of the antibody will not be present. Under normal circumstances, however, the isolated antibody is prepared by at least one purification step.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the identity of the antibody obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention can be prepared by methods described in Kohler et al, 1975, Nature 256: 495 or may be prepared by recombinant DNA methods (see, e.g., U.S. patent No.4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described in the following references: clackson et al, 1991, Nature 352: 624-: 581-597. Monoclonal antibodies herein expressly include "chimeric" antibodies.

Monoclonal antibodies can be prepared according to the knowledge and techniques of the art: the human APRIL antigen is injected into a test subject and then hybridomas expressing antibodies with the desired sequence or functional characteristics are produced. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA.

APRIL-binding peptides obtained with the process of the invention for producing APRIL-binding peptides (e.g., antibodies or analogs thereof) may comprise immunoglobulin V_HDomain of an immunoglobulin V_HThe domains comprise amino acid sequences that are identical to the amino acid sequences respectively selected from SEQ ID NOs: 5. 6 and 7, or SEQ ID NO: 15. 16 and 17, or SEQ ID NO: 25. 26 and 27, or SEQ ID NOs: 35. 36 and 37, or SEQ ID NOs: 45. 46 and 47 have a CDR1, CDR2 and CDR3 sequence having at least 60%, such as at least 85%, preferably at least 90%, more preferably at least 95% sequence similarity, such as V_HThe domain has at least 60%, such as at least 85%, preferably at least 90%, more preferably at least 95% sequence similarity to an amino acid sequence selected from the group consisting of SEQ ID No.3, 13, 23, 33 or 43.

The APRIL-binding peptide (e.g., an anti-APRIL antibody or analog thereof) may comprise an immunoglobulin V_HAnd V_LDomain of an immunoglobulin V_HAnd V_LThe domains comprise amino acid sequences that are identical to the amino acid sequences respectively selected from SEQ ID NOs: 5. 6,7, 8, 9 and 10, or SEQ ID NO: 15. 16, 17, 18, 19 and 20, or SEQ ID NO: 25. 26, 27, 28, 29 and 30, or SEQ ID NO: 35. 36, 37, 38, 39 and 40, or SEQ ID NO: 45. 46, 47, 48, 49 and 50 has a V with a sequence similarity of at least 60%, such as at least 85%, preferably at least 90%, more preferably at least 95%_HCDR1、V_HCDR2、V_HCDR3、V_LCDR1、V_LCDR2 and V_LCDR3 sequences, e.g. V_HAnd V_LA pair of domains with a pair of domains selected from SEQ ID NOs: 3 and 4, or 13 and 14, or 23 and 24, or 33 and 34, or 43 and 44, have a sequence similarity of at least 60%, such as at least 85%, preferably at least 90%, more preferably at least 95%. The coding DNA sequences of these various sequences can be determined by the skilled person based on his knowledge of the genetic code. In Table 2 below, a number of V's are listed_HAnd V_LMany coding DNA sequences of amino acid sequences. The sequences are provided in the sequence listing.

The APRIL-binding peptides of the invention find use as diagnostic and/or analytical tools, preferably for use in ex vivo diagnostic methods. Accordingly, other aspects of the invention relate to such use of APRIL-binding peptides. For example, APRIL-binding peptides may be used to detect APRIL in a sample from a subject, such as a tissue sample, whole blood, or a blood-derived sample (e.g., plasma or serum). Alternatively, APRIL-binding peptides can be used to detect APRIL on specific cells (e.g., cells derived from tissue, blood, or culture). Such tests are intended to diagnose conditions associated with altered levels of APRIL, such as conditions selected from cancer, conditions associated with inflammation, sepsis, allergy, autoimmune diseases or infections (e.g., bacteremia). The test is used in determining whether a subject has a diagnosis of a condition associated with altered levels of APRIL. In this case, it is generally assessed whether the subject has an elevated (higher than normal) APRIL level. The current standard for normal human serum APRIL levels is about 1-10ng/ml (Planelles et al, 2007, Haematologica 92, 1284-5). Thus, in the present invention, the altered level of APRIL may be an elevated level of APRIL, such as an APRIL level above 10ng/ml, such as above 15, 30, 50 or 100 ng/ml. Alternatively, where a normal APRIL level is known for a particular subject (e.g., from multiple determinations made at multiple specific time points deemed to be associated with a normal APRIL level), an elevated APRIL level can be determined relative to a normal APRIL level determined for the subject. Alternatively, the test may be used to assess the outcome of treatment in which a subject receiving treatment suffers from a condition associated with altered levels of APRIL and/or stabilizes it. In this case, it is generally assessed whether elevated APRIL levels in the subject are reduced or closer to what is considered normal. It is evident that after a subject has been specifically diagnosed as having a condition associated with altered levels of APRIL, in particular elevated levels of APRIL, the diagnosis may proceed to evaluate the subject's acceptance of treatment for and/or stabilization of the condition associated with altered levels of APRIL.

Cancers that may benefit from assays using APRIL-binding peptides of the invention may be selected from leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic promyelocytic leukemia, myelomonocytic erythroleukemia, chronic leukemia, chronic myelogenous (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, burkitt's lymphoma and marginal zone B-cell lymphoma, erythrocytosis lymphoma, hodgkin's disease, non-hodgkin's disease, multiple myeloma, waddenston's macroglobulinemia, heavy chain disease, solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioleiiosarcoma, synovioma, mesothelioma, lymphoblastoma, lymphoblastoid, lymphoblastic lymphoma, lymphoblastic leukemia, and lymphoblastic lymphoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, bone marrow carcinoma, bronchial cancer, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonic cancer, Wilm's tumor, neck cancer, uterine cancer, testicular tumor, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal cancer, basal cell carcinoma, biliary tract cancer, squamous cell carcinoma, neuroblastoma, melanoma, neuroblastoma, melanoma, and esophageal cancer, Bladder cancer, bone cancer, brain and Central Nervous System (CNS) cancer, neck cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial tumors, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, and cancer of the urinary system.

Conditions associated with inflammation that may benefit from assays using APRIL-binding peptides of the invention are atherosclerosis, acne vulgaris, celiac disease, chronic prostatitis, glomerulonephritis, allergy, reperfusion injury, sarcoidosis, rejection inhibition, vasculitis, interstitial cystitis, and conditions associated with aseptic inflammation (including muckle-weidi syndrome and other autoinflammatory disorders).

Another condition that may benefit from assays using APRIL binding peptides of the invention is sepsis and/or related conditions, such as Systemic Inflammatory Response Syndrome (SIRS). The pathology of sepsis is known to the skilled person. In particular, the skilled person understands that sepsis can be defined as an infection-induced syndrome involving two or more of the following features of systemic inflammation: fever or hypothermia, leukocytosis or leukopenia, tachycardia and tachypnea or hyperventilation. The skilled person is also aware that the development of sepsis in a subject may undergo such a process, development: from systemic inflammatory response syndrome ("SIRS") -negative to SIRS-positive, then to sepsis, which can then progress to severe sepsis, septic shock, multiple organ dysfunction ("MOD"), and ultimately death. Sepsis can also occur in infected subjects when they subsequently develop SIRS. Such "sepsis" can be defined as a systemic host response to an infection that is documented as SIRS infection. "severe sepsis" is associated with MOD, hypotension, disseminated intravascular coagulation ("DIC") or hypoperfusion abnormalities, including lactic acidosis, oliguria and changes in mental status. "septic shock" is generally defined as sepsis-induced hypotension that resists fluid resuscitation due to the additional presence of hypoperfusion abnormalities.

Autoimmune diseases that may benefit from assays using APRIL-binding peptides of the invention may be selected from multiple sclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, crohn's disease and other inflammatory bowel diseases such as ulcerative colitis, Systemic Lupus Erythematosus (SLE), autoimmune encephalomyelitis, Myasthenia Gravis (MG), hashimoto's thyroiditis, goodpasture's syndrome, pemphigus, glaff's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-antigen antibodies, mixed connective tissue disease, polymyositis, malignant disease, idiopathic addison disease, autoimmune-related infertility, glomerulonephritis crescentis, proliferative glomerulonephritis, bullous pemphigoid, sjogren's syndrome, psoriatic arthritis, insulin resistance, and the like, Autoimmune diabetes, autoimmune hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome, autoimmune glucose retinitis, Guillain-Barre syndrome, atherosclerotic disease, and Alzheimer's disease.

Testing exemplary allergic conditions using APRIL-binding peptides of the present invention may include, but are not limited to, allergic conjunctivitis, vernal keratoconjunctivitis, and giant papillary conjunctivitis; nasal allergic disorders including allergic rhinitis and sinusitis; ear allergic disorders including eustachian tube pruritus; allergic disorders of the upper and lower respiratory tract, including intrinsic and extrinsic asthma; allergic disorders of the skin including dermatitis, eczema and urticaria; and allergic disorders of the gastrointestinal tract.

Some examples of pathogenic viruses that cause infection include HIV, hepatitis virus (A, B, C, D or E), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II and CMV, EB (Epstein Barr) virus), adenovirus, influenza virus, arbovirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, polio virus, rabies virus, JC virus arbovirus encephalitis virus.

Some examples of pathogenic bacteria that cause infection include chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumococci (pneumococci), meningococci and gonococci (conocci), klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera bacteria, tetanus, botulirus, anthrax bacteria, plague bacteria, leptospirosis bacteria, and lyme disease bacteria.

Some examples of pathogenic fungi that cause infections include Candida (Candida), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucor (Genus Mucorales), Mucor, Rhizopus, Sporotrichii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum (Histoplasma capsulatum).

Some examples of pathogenic parasites causing infection include entamoeba histolytica (Entamoebahistolytica), Enterobacter coli (Balatidium coli), Engler's Neurospora Fowleri (Naegleriafarleri), Acanthamoeba species (Acanthamoeba sp.), Giardia lamblia (Giardia lambia), Cryptosporidium species (Cryptosporidium sp.), Pneumocystis carinii (Pneumocystis carinii), Plasmodium vivax (Plasmodivivax), Babesia cubensis (Babesia micoti), Trypanosoma brucei (Trypanosoma brucei), Trypanosoma cruzi (Trypanosoma cruzi), Leishmania donii (Leishhnonovanini), Toxoplasma gondii and Trichoderma reesei (Nitrosomus).

For diagnostic applications, the APRIL-binding peptides of the invention are typically linked (directly or indirectly) to a detectable label group, a signaling moiety. A number of label portions are available, which can be grouped generally into the following categories: biotin, fluorescent dyes, radionucleotides, enzymes, iodine and biosynthetic markers. In addition, in the case where the APRIL-binding peptide is an antibody or an analog thereof, the Fc chain may serve as a labeling moiety. Also, an Fc chain may be added to the non-antibody binding peptide as a label. There are many antibodies on the market that target Fc chains from various species (e.g., anti-murine and anti-human antibodies). These antibodies are available in the form of various labels, and they can be used in known methods to target the Fc chain of the anti-APRIL antibodies of the invention. Thus, where an anti-APRIL antibody is used, it is preferably an anti-APRIL antibody that is antigenically distinct from other proteins present in the assay (e.g., by at least predominantly comprising protein sequences from a heterologous source), such as APRIL and APRIL receptor (or binding equivalents thereof). This facilitates targeting of the Fc-chain of the anti-APRIL antibody with the labeled antibody during the detection process. The skilled person will appreciate that for chimeric antibodies, the Fc chain primarily determines the antigenicity of the antibody.

According to certain embodiments of the test methods, an APRIL-binding peptide is detected in a sample from a subject, and the presence of the APRIL-binding peptide is used as an indicator of the presence of APRIL. In these embodiments, the APRIL-binding peptide is typically used in a soluble form. According to certain other embodiments of the assay method, the APRIL-binding peptide is immobilized on a solid support and used as a capture agent to capture APRIL or a complex comprising APRIL.

For example, in a first test mode, APRIL-binding receptors (such as BCMA or TACI) or binding equivalents thereof (such as hapril.01a (disclosed in WO2010/100056) or analogs thereof) can be immobilized on a solid support and a sample (e.g., serum) from a subject applied to the solid support. After washing (to remove unbound material, in particular unbound material that interferes with the detection of APRIL), APRIL-binding peptide is added to the solid support and the presence of APRIL-binding peptide is detected, for example, by detecting a label attached to the APRIL-binding peptide or, according to certain embodiments, by adding a labeled antibody specific for the Fc chain of the APRIL-binding peptide. The detection may be qualitative, semi-quantitative or quantitative. Quantitative detection is preferred. Methods and means for detecting labeled peptides, such as labeled antibodies, are known to the skilled artisan. For example, use may consist of a horseradish peroxidase conjugated antibody that binds to the Fc chain of an APRIL-binding antibody. The conversion of revealing substrates (e.g., TMB, DAB, ABTS) to colored products by horseradish peroxidase was used as a measure of bound APRIL.

In an alternative diagnostic mode, the diagnostic test is for detecting the amount of APRIL in a sample from the subject that forms a complex with an APRIL receptor equivalent (e.g., hapril.01a or an analog thereof) administered to the subject. In this mode, the assay may comprise:

-providing an APRIL-binding peptide immobilized on a solid support;

-applying the sample to a solid support and incubating to allow complexes present in the sample to bind to the solid support;

-washing;

-detecting bound complexes and/or detecting bound uncomplexed APRIL.

This test mode is valuable, for example, in determining the saturation of APRIL in a subject's serum by blockers of the APRIL-APRIL receptor interactors. For this purpose, bound complexes are preferentially detected and bound uncomplexed APRIL is detected. This can be achieved by: complexed and uncomplexed APRIL are detected in separate incubations and/or different labels are used when targeting the bound complex and the bound uncomplexed APRIL. Uncomplexed APRIL can also be determined in the first test mode described above.

To detect bound complexed APRIL and bound uncomplexed APRIL in the same incubation, the complex can be targeted with a first detection peptide (e.g., an antibody) directed against the APRIL receptor equivalent, which is bound to a first label, and uncomplexed APRIL can be targeted by a second detection peptide (e.g., an antibody) directed against APRIL, which is bound to a second label that is different from the first label. A second test peptide may be selected that binds to the same APRIL region as the APRIL receptor equivalent. It may therefore be selected as the APRIL receptor or an APRIL receptor equivalent. Indeed, in its binding to APRIL, it is equivalent to the receptor equivalent administered to a subject. Thus, the second detection peptide can be slightly different from the receptor equivalent administered to the subject by containing a linking label, such as hapril.01a or an analog thereof, that allows for detection differentiation from the detection of the receptor equivalent administered to the subject. For example by containing different Fc-chains. In the present invention, reference to hapril.01a includes analogs thereof, particularly humanized analogs. Clearly, the second detection peptide should preferably not bind to the same region of APRIL, wherein the APRIL-binding peptide selected binds to APRIL and its binding to APRIL should also not be blocked by the binding of the APRIL-binding peptide selected.

The assay results in detection of bound complex and/or bound uncomplexed APRIL, as detected. From the results of the assay, the level of saturation of APRIL by administration of an APRIL receptor equivalent can be determined, allowing assessment of the therapeutic outcome of treatment of a subject with an APRIL receptor equivalent (e.g., hapril.01a or an analog thereof).

In an embodiment of the diagnostic assay of the invention, the APRIL-binding peptide or APRIL-binding receptor (or binding equivalent thereof) may be immobilized, for example, on the surface of a laboratory container, such as the surface of a microtiter well plate. Clearly, by applying the sample to the solid support, the sample is applied to the immobilized APRIL-binding peptide.

Washing is to remove unbound material, in particular unbound material that interferes with the APRIL assay, and can be accomplished with any suitable washing solution known to the skilled person. Aqueous solutions are generally used. Certain general guidance regarding washing solutions is also provided above regarding methods of obtaining APRIL-binding peptides.

It is clear that in the diagnostic test of the present invention reactions and processes such as the following can be carried out in suitable containers, such as reactors, in particular vessels used in laboratory scale for diagnostic purposes: applying the sample to a solid support and incubating to allow complexes present in the sample to bind to the solid support, a washing step and a detection step.

The exemplary diagnostic modalities described above involve a solid support with immobilized peptides. It is clear to the skilled person that the diagnostic test mode can also be carried out entirely in solution, for example by using a nuclear magnetic resonance energy transfer (FRET) methodology, including time-resolved FRET (TR-FRET or HTRF). The skilled person knows how to modify the assay format described above in order to perform it completely in solution, for example by using FRET methodology. Furthermore, the APRIL-binding peptides of the present invention are beneficial in assay formats that are fully performed in solution, as they can be used as indicators of the presence of APRIL.

The skilled artisan understands that in various assay formats, reduced interference of APRIL-binding peptides with APRIL-APRIL receptor interactors is beneficial. However, it should be emphasized that the diagnostic use of the peptides of the invention is not limited to these exemplary test modes. The potential of APRIL-binding peptides of the invention for use in diagnostic and analytical applications is further supported by the results shown in the experimental section.

The APRIL-binding peptides of the invention may also be used in any other known assay such as competitive binding assays, direct and indirect sandwich assays and immunoprecipitation assays (Zola, Monoclonal antibodies. a manual of Techniques, page 147-158(CRC Press, inc. 1987)).

The APRIL-binding peptides of the present invention may also be used in vivo diagnostic assays. Typically, the APRIL-binding peptide is labeled with a radionuclide so that the APRIL antigen or cells expressing the APRIL-binding peptide can be localized using immunoscintigraphy or positron emission tomography.

The APRIL-binding peptides of the present invention may also have other non-therapeutic uses. Non-therapeutic uses of APRIL-binding peptides include flow cytometry, western blotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry.

The APRIL-binding peptides of the invention may also be immobilized, for example, on a protein a agarose column and used as affinity purifiers.

The invention is further illustrated by reference to the following examples, which show non-limiting embodiments of the invention.

Examples

Example 1

Commercially available APRIL detection assays do not reliably detect APRIL in Human Serum (HS)

For the detection of APRIL in patient serum, ELISA-based assays have been described which rely on capturing APRIL by human BCMA and detecting bound antibodies using polyclonal rabbit APRIL-specific antibodies (Planelles L et al, Haematologica 2007, 92: 1284-5). However, polyclonal antibodies have limited availability and are not reproducibly available. To address this issue, commercially available anti-APRIL assays were compared to the detection observed with polyclonal antibody-based ELISA. For the polyclonal ELISA, plates were coated with BCMA-Fc (R & D systems) in 100 ng/well coating buffer (0.2M sodium phosphate buffer, pH 6,5) at 4 ℃. After overnight coating, plates were washed three times with PBS plus 0.05% tween 20 (PBST). Plates were then blocked with PBS containing 10% human serum (assay diluent) for one hour at room temperature. For commercial ELISA, pre-coated plates were used (Biolegged, San Diego, USA). Standard curves were generated in assay dilutions using recombinant human APRIL (R & Dsystems) or using supplied recombinant human APRIL (biolegend). Subsequently, human serum from colorectal cancer patients was diluted ten-fold with assay diluent and tested in parallel in both ELISAs. Detection of bound APRIL was then performed with either the monoclonal antibody provided coupled to horseradish peroxidase or with polyclonal antibodies and subsequently by a second step using goat-anti-rabbit peroxidase (diluted 1:1,000 in assay diluent) according to the manufacturer's instructions (Biolegend). Antibody incubation steps were all performed at room temperature for one hour in assay dilution and then washed three times with PBS/0, 05% tween 20.

As shown in fig. 1, the results of the polyclonal antibody based assay are not reproducible by commercially available APRIL ELISA. In several instances, high detection was observed with the polyclonal ELISA, which did not match the high expression in the Biolegend ELISA. In contrast, several examples showed significant detection by Biolegend ELISA, whereas APRIL was not detected in the polyclonal ELISA. Correlation analysis using spearman R values indicated a very low correlation 0,5946, with relatively poor confidence intervals. Thus, ELISA does not provide comparable data.

In addition, the effect of the addition of human serum on APRIL was determined to analyze the effect of human serum on the quantification of APRIL. Two standard curves were generated using recombinant APRIL generated by transfecting APRIL-expressing constructs into 293T cells. The recombinant APRIL was diluted with PBS + 10% fetal bovine serum + 20% Human Serum (HS) (Sigma, cat num H4522) or with PBS/1% BSA at concentrations of 100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.136 and 0.04 ng/ml. The binding of these two standard curves was tested based on several commercially available antibodies or ELISA kits to determine the effect of serum addition on APRIL quantification.

For APRIL ELISA provided by the Biolegend, Legend MAX ELISA kit (cat number: 439307) with pre-coated plates, all reagents were placed in room temperature prior to use. By using eachThe plate was washed 4 times with at least 300 μ l of 1 × wash buffer (designated by the manufacturer) and any residual buffer was blotted by tapping the plate firmly inverted on the adsorption paper. Next, 100 μ l of standard curve diluent was added, the plate was sealed with a blocking layer (plateau seal) contained in the kit, and incubated at room temperature for 2 hours while shaking at 200 rpm. After this step, the plate was washed four times with 1 × wash buffer. Next, 100 μ l of human APRIL/TNFSF13 detection antibody solution (designated by the manufacturer) was added to each well, sealed and incubated at room temperature for 1 hour while shaking at 200 rpm. After incubation, the plates were washed four times with 1 × wash buffer. Primary antibodies were recognized by adding 100 μ l avidin-HRP a solution (designated by the manufacturer) to each well and incubated for 30 minutes at room temperature while shaking. Finally, the plate was washed five times with 1X wash buffer. The binding complexes were visualized by adding 100 μ l of substrate solution F (specified by the manufacturer) to each well and incubating for 15 minutes in the dark at room temperature. The reaction was stopped by adding 100. mu.l of stop solution (designated by the manufacturer) to each well. Absorbance was read at 450 nm. Figure 2A shows the APRIL assay decreased in this assay for the presence of human serum. Similar to the polyclonal antibody assay described above, the detection of APRIL by BCMA-Fc capture followed by the use of commercially available monoclonal antibodies was evaluated. In FIG. 2B, the effect of human serum on the detection of APRIL is presented using APRILY-5 as the detection monoclonal antibody. With 100. mu.l of 0.5. mu.g/ml BCMA-Fc in coating buffer (EBC 0512081; R)&D) ELISA plates were coated (see above) and incubated overnight at 4 ℃. Plates were washed three times with PBS/0.2% Tween (as described previously) and blocked with 150. mu.l PBS/1% BSA at 37 ℃ for one hour. After three washes with PBS/0.2% tween, 100 μ l of different standard curve solutions were added to each well. The standard curve concentration was incubated at room temperature for 2 hours. After the incubation time, plates were washed three times with PBS/0.2% tween. Next, 100. mu.l of commercially available biotinylated APRILY-5(ALX-804-801-C100, Enzo Life Sciences BVBA, Antwerpen, Belgium) was diluted at 1. mu.g/ml in PBS/1% BSA and incubated for one hour at 37 ℃. Next, plates were washed three times with PBS/0.2% tween. The next stepIncludes the addition of 100. mu.l streptavidin-HRP (cat. number890803, R) diluted at 1:1,000 in PBS/1% BSA&D, UK), incubated at 37 ℃ for one hour. Plates were washed three times with PBS/0.2% tween and bound immune complexes were visualized using 100 μ l TMB substrate. Using 100. mu.l of 0.5M H₂S0₄The reaction was stopped and the absorbance read at 450 nm. APRIL was not detected in the presence of human serum.

In fig. 2C, the effect of human serum on the quantification of APRIL was determined using an ELISA assay using commercially available sasca-2 anti-APRIL antibodies to capture APRIL and APRILY-5 biological antibodies to detect bound APRIL. In this assay, ELISA plates were coated with 100. mu.l of anti-APRIL antibody Sascha-2 (804-C100, Enzo LifeSciences BVBA, Antwerpen, Belgium) in coating buffer (see above) and incubated overnight at 4 ℃. Plates were washed three times with PBS/0.2% Tween and blocked with 150. mu.l PBS/1% BSA for one hour at 37 ℃. After three washes with PBS/0.2% Tween, 100. mu.l of standard curve was incubated. 100 μ l of this standard curve solution was added to each well and the standard curve concentration was incubated at room temperature for two hours. After incubation, plates were washed three times with PBS/0.2% tween. Next, 100. mu.l of commercially available biotinylated APRILY-5 was added to the plate at a concentration of 1. mu.g/ml diluted in PBS/1% BSA and incubated for one hour at 37 ℃. Next, plates were washed three times with PBS/0.2% tween. The next step included adding 100. mu.l of streptavidin-HRP diluted 1:1,000 in PBS/1% BSA and incubating for one hour at 37 ℃. Plates were washed 3 times with PBS/0.2% tween and bound immune complexes were visualized by adding 100 μ l TMB substrate. Using 100. mu.l of 0.5M H₂S0₄The reaction was stopped and the absorbance read at 450 nm. Unreliable detection of APRIL was observed in the absence or presence of human serum.

Finally, in FIG. 2D, we evaluated the use of a second commercially available APRIL ELISA, DuoSet ELISA anti-human APRIL/TNFSF13(cat num DY884) from R & D Systems. Plates were coated by diluting the capture antibody (designated by the manufacturer, 843362) to a final concentration of 2 μ g/ml with PBS. 96-well plates were coated at 100. mu.l per well and incubated overnight at room temperature. The next day, plates were washed four times with wash buffer (cat num WA126, specified by the manufacturer). Plates were blocked by adding 300 μ l reagent dilution (designated by manufacturer, cat num DY995) to each well and incubated for 1 hour at room temperature. The plate was then washed four times. Later, 100 μ l of standard curve fluid or serum samples were added. The serum samples were diluted five times with reagent dilutions, covered with adhesive strips and incubated for 2 hours at room temperature. Next, the plates were washed four times, 100 μ l of detection antibody (cat num 843363, specified by the manufacturer) diluted with reagent diluent was added to each well, and APRIL in serum was detected by incubation at room temperature for 2 hours. The plate was washed four times. Then, 100 μ l of working dilution streptavidin-HRP (cat num 890803, R & D, UK) was added to each well, the plates were covered and incubated for 20 minutes at room temperature. The plate was washed four times. Immune complexes were visualized by adding 100 μ l of substrate solution (designated by manufacturer, DY999) to each well and incubating for 20 min at room temperature. The reaction was stopped by adding 50. mu.l of stop buffer (DY994) to each well. Bound APRIL was detected by optical density at 450 nm. No APRIL was detected.

In summary, none of the commercially available ELISA assays or monoclonal anti-APRIL antibodies reproduced the assay results obtained with polyclonal antibodies as described above, and all of these demonstrated a large interference of human serum with the quantification of human APRIL.

Example 2

Immunization and selection of anti-APRIL antibodies

Immunization of mice with APRIL cDNA

To isolate antibodies against human APRIL protein and allowing detection of APRIL in the context of human serum, mice were immunized with hAPRIL cDNA. Second, selection procedures were designed and developed to specifically isolate cells expressing anti-hAPRIL antibodies that bind human APRIL that interacts with BCMA binding.

anti-hAPIL antibodies were raised by cDNA immunization of mice. First, cDNA encoding the full-length open reading frame of hAPRIL was subcloned into the pCI-neo vector (Promega, Madison, Wis.). The expression of the obtained vector was examined by the following steps: pCI-neo-hAPRIL was transiently transfected into 293 cells (American Type Culture Collection, Manassas, Va.) and immunoblotted with mouse anti-hAPRIL IgG1 April-5 (1: 5,000) (Alexis, San Diego, Calif.) followed by goat anti-mouse IgG1-HRP (1: 2,000) (Southern Bio technology, Birmingham, AL). Mice were immunized by biolistic immunization using a Helios gene gun (BioRad, Hercules, CA) and DNA-coated gold bullets (BioRad) according to the manufacturer's instructions. Briefly, the reaction was carried out with 2: 1:1 ratio of pCI-neo-hAPRIL cDNA and mouse Flt3L commercial expression vector vectors and mouse GM-CSF (both from Aldevron, Fargo, ND) coated 1 μm gold particles. A total of 1. mu.g of plasmid DNA was used to coat 500. mu.g of gold particles.

Specifically, 7-8 week adult female BALB/C mice were immunized with the gene gun in the ears, which received 3 cycles of bombardment in both ears. Approximately 1: 800-2,400 anti-hAPIL titers. In ELISA, after all incubation steps, a washing step of PBST (PBS with 0.01% tween 20) was performed. Maxisorp96 well immunoplates (Nunc, Rochester, NY) were coated overnight at 4 ℃ with rabbit anti-FLAG polyclonal antibody (50 ng/well PBS) (Sigma, F7425) and blocked with 10% goat serum/PBST for 1 hour at room temperature. Plates were incubated with supernatant (1:10PBS) from 293T cells transiently transfected with a CMV promoter promoting secreted form of FLAG-hAPRIL (pCR3-hAPRIL) for 1 hour at room temperature, followed by dilution with mouse serum and 1: 2,000HRP enzyme-labeled goat anti-mouse IgG (southern Biotechnology) was incubated at room temperature for 1 hour, respectively. After the final PBST wash, anti-hAPRIL immunoreactivity was visualized with 100 μ l of stabilized chromagen (Invitrogen, SB 02). Using 100. mu.l of 0.5M H₂S0₄The reaction was stopped and absorbance was read at 450nm and 620 nm. Finally, mice that exhibited a response to hAPRIL were immunized a third time and sacrificed four days later.

Red blood cell depleted spleen and lymph node cell populations were prepared as previously described (Steenbakkers et al, 1992, J.Immunol.meth.152: 69-77; Steenbakkers et al, 1994, mol.biol.Rep.19: 125-134) and frozen at-140 ℃.

Selection of anti-APRIL antibody-producing B cells

In order to specifically select B cells that produce anti-hAPRIL antibodies that detect APRIL in the presence of human serum, a selection strategy was designed and developed that: it preferably binds to B cells expressing an anti-hAPRIL antibody that binds to APRIL that binds BCMA-Fc (fig. 3). 20 μ g of recombinant BCMA-Fc (R) in 0.1M phosphate buffer (pH 7.4) was used&D systems, cat #193-13C) 4X10 at 4 deg.C⁷M-450 tosyl activated magnetic beads (magneticDynabeads) (Cat 140.13) were incubated throughout the weekend. Second, the supernatant was aspirated and the beads were blocked by incubation with PBS/1% BSA for one hour at 4 ℃. Next, the beads were washed 3 times with PBS/0.1% BSA. Subsequently, the beads were incubated with FLAG-APRIL containing supernatant (1:10PBS from 293T cells transiently transfected with a CMV promoter promoting secreted form of FLAG-hAPIL (pCR 3-hAPIL)) by incubation at 4 ℃ for one hour. Finally, the beads were resuspended in PBS/0.1% BSA.

To select B cell clones producing blocking-reducing anti-hAPIL antibodies, 1.4X10 was used⁷The spleen cells depleted of erythrocytes were thawed. hAPRIL-specific B cells were selected by selection of splenocytes based on APRIL-BCMA complexed with tosyl activated magnetic beads in a bead to cell ratio of 1: 1.5. Non-specifically bound splenocytes were washed away by 15 Xwash with 5ml of DMEMF 12/P/S/10% BCS medium. Secondly, the ratio of the average molecular weight of the polymer is determined according to Steenbakkers et al, 1994, mol.biol.Rep.19: 125-134 the selected B cells were cultured. Briefly, selected B cells were mixed with 7.5% (v/v) T cell supernatant and 50,000 irradiated (2,500RAD) EL-4B5 feeder cells in a final volume of 200. mu.l of DMEMF 12/P/S/10% BCS in 96-well flat-bottom tissue culture plates.

On day nine, supernatants were screened by ELISA based on hAPRIL reactivity. In ELISA, all incubation steps were followed by a washing step of PBST (PBS with 0.01% tween 20). With 0.2. mu.g/ml BCMA-Fc (R) in PBS&D Systems, 193-13C) Maxisorp 96-well immunoplates (Nunc, Rochester, NY) (50. mu.l/well PBS) were coated overnight at 4 ℃ and blocked with PBS/1% BSA at room temperature for 1 hour. With promoters from transiently transfected CMVThe supernatant of 293T cells secreting FLAG-hAPRIL (pCR3-hAPRIL) in the form of a plasmid (1:10PBS) was incubated for 1 hour at room temperature, followed by 1 hour at room temperature with 50. mu.l of supernatant from B cell culture medium and 1:5,000HRP enzyme labeled goat anti-mouse IgG (southern Biotechnology). After the final PBST wash, anti-hAPRIL immunoreactivity was visualized with 100. mu.l Stabilizedchromagen (Invitrogen, SB 02). Using 100. mu.l of 0.5M H₂S0₄The reaction was stopped and the absorbance read at 450 nm. B cell clones expressing hAPRIL-reactive antibodies were identified by ELISA.

Subsequently, B cell clones from the supernatant of the hAPRIL reaction were immortalized by electrofusion using the following published procedure (Steenbakkers et al, 1992, J.Immunol.meth.152: 69-77; Steenbakkers et al, 1994, mol.biol.Rep.19: 125-34). Specifically, the B cells were combined with 10⁶Sp2/0-Ag14 myeloma cells were mixed and the serum was removed by washing with DMEM F12 medium. Cells were treated with pronase solution (Calbiochem, cat. No.4308070.536) for 3 min and washed with electrofusion equimolar buffer (Eppendorf, cat. No. 53702). Electrofusion was performed in a 50. mu.l fusion chamber by an alternating electric field of 30s, 2MHz, 400V/cm, followed by a square high electric field pulse of 10. mu.s, 3kV/cm and again an alternating electric field of 30s, 2MHz, 400V/cm.

The contents of the fusion chamber were transferred to hybridoma selection medium and placed in 96-well plates under limited dilution conditions. Hybridoma supernatants were screened on day 12 post-fusion based on hAPRIL binding activity, as described above. Five hybridomas secreting antibodies recognizing hAPRIL in the supernatant were subcloned by limited dilution to protect their integrity. The following anti-hAPRIL antibodies were selected for further analysis: hAPRIL.130, hAPRIL.132, hAPRIL.133, hAPRIL.135, hAPRIL.138.

The selection strategy used to identify APRIL-binding peptides, here immunoglobulins expressed on B cells (B), is shown schematically in fig. 3. In this schematic, BCMA-Fc (as a shielding peptide) is bound (or otherwise immobilized) to a solid support (bead), and the target peptide (APRIL) is immobilized on the solid support by interaction with BCMA. However, it is clear from the above description that: in alternative embodiments, the target peptide may be bound (or otherwise immobilized) to a solid support, and the shielding peptide may be immobilized on the solid support through its interaction with the target peptide.

Example 3

Purification and characterization of anti-APRIL antibodies

Stability of anti-APRIL producing hybridomas and purification of anti-APRIL antibodies

Clonal populations of hAPRIL hybridomas were obtained by two limited dilutions. Culturing the stable hybridoma in a serum-free culture medium for 7-10 days; the supernatant was collected and filtered through a 0.22 μ M nitrocellulose membrane. Antibodies were purified using mAb Select SuReProtA resin according to the manufacturer's instructions (GE Healthcare, cat. No. 17-5438). When a PD-10 gel filtration column (GE Healthcare) was used, the buffer was changed to PBS. Antibodies were quantified using spectrophotometry. All (sub) -isotypes of hAPRIL antibodies were determined as IgG1, κ using the mouse monoclonal antibody isotype test kit (Roche, # 11493027001).

Cloning of immunoglobulin cDNA

Degenerate primer-based PCR methods were used to determine the expression of the protein encoded by hAPRIL hybridomas: the DNA sequence of the variable region of the mouse antibodies expressed hAPRIL.130, hAPRIL.132, hAPRIL.133, hAPRIL.135 and hAPRIL.138.

RNeasy Mini kit (Qiagen, 74106) was used from about 5X10 according to the manufacturer's instructions⁶Total RNA was isolated from individual hybridoma cells and treated with dnase i (invitrogen) according to the manufacturer's instructions. Gene-specific cDNAs for the heavy and light chains were synthesized using M-MLV reverse transcriptase, RNase H-, point mutant kit (Promega, cat. NO. M3683) according to the manufacturer's instructions. V was amplified using Novagen-based Ig primer set (Novagen, San Diego, Calif.) and Accuprime Pfx DNA polymerase (Invitrogen)_HAnd V_LThe gene was amplified by PCR. All PCR products matching the expected amplicon size of 500bp were cloned into pCR4TOPO vector (Invitrogen) and the constructs were transformed into the pCR4TOPO vector according to the manufacturer's instructionsSubcloned high Performance DH5 α competent cells (Invitrogen).

Clones were screened by colony PCR using universal M13 forward and reverse primers, and at least two clones from each reaction were selected for DNA sequencing analysis. CDRs were identified according to Kabat's rules (Kabat et al, 1991.Sequences of proteins of Immunological Interest, fifth edition, NIH issue Nos. 91-3242).

The sequences are disclosed in the accompanying sequence listing and are listed in table 1 above.

Example 4

Detection of APRIL in human and transgenic mouse serum by anti-APRIL antibodies

To quantify APRIL serum levels in serum derived from CLL patients, APRIL monoclonal antibodies that were just identified were used: hAPRIL.130, hAPRIL.132, hAPRIL.133, hAPRIL.135 and hAPRIL.138, were analyzed by ELISA as follows. The ELISA plates were coated with 100. mu.l of 0.5. mu.g/ml BCMA-Fc (EBC 0512081; R & D) in coating buffer (0,2M sodium phosphate, pH 6,5) and incubated overnight at 4 ℃. Next, plates were washed three times with PBS/0.2% Tween and blocked with 150. mu.l PBS/1% BSA for one hour at 37 ℃. After washing 3 times with PBS/0.2% Tween, 100. mu.l of the sample or standard curve was added. CLL serum samples were diluted five-fold with PBS/10% FCS, while standard curves were diluted with PBS + FCS 10% + HS 20%. The samples and standard curve concentrations were incubated at room temperature for 2 hours. Next, plates were washed three times with PBS/0.2% tween. Next, 100. mu.l of anti-APRIL monoclonal antibody was added to the plate at a concentration of 1. mu.g/ml diluted in PBS/1% BSA and incubated at 37 ℃ for one hour. Subsequently, plates were washed three times with PBS/0.2% tween, and 100 μ l of a detergent was added at 1:1,000 goat anti-mouse IgG (H & L) (Southern Biotech, cat number 1031-05) diluted in PBS/1% BSA and incubated for one hour at 37 ℃. Plates were washed three times with PBS/0.2% tween and bound immune complexes were visualized by addition of 100 μ Ι TMB substrate (TMB). The reaction was stopped by adding an equal amount of 1M hydrochloric acid to the reaction volume. Binding to APRIL was quantified by measuring optical density at 450 nm. As shown in fig. 4A, all monoclonal antibodies revealed APRIL in the patient's serum to the same extent.

Next, the assay was extended using the hapril.133 monoclonal antibody using the same ELISA setup. Additional samples of 10 CLL patients with different amounts of APRIL were analyzed (fig. 4B).

In addition, the effect of the presence of human serum on the quantification of APRIL was investigated using an assay format that utilizes BCMA-Fc capture and hapril.133 antibody for detection. Two standard curves were generated using recombinant APRIL generated by transfection of APRIL-expressing constructs into 293T cells. The recombinant APRIL was diluted with PBS + 10% fetal bovine serum + 20% Human Serum (HS) (Sigma, cat number H4522) or PBS/1% BSA at concentrations of 100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.136 and 0.04 ng/ml. The binding of these two standard curves was determined (FIG. 5). The ELISA plates were coated with 100. mu.l of 0.5. mu.g/ml BCMA-Fc (EBC 0512081; R & D) in coating buffer (0,2M sodium phosphate, pH 6,5) and incubated overnight at 4 ℃. Next, plates were washed three times with PBS/0.2% Tween and blocked with 150. mu.l PBS/1% BSA at 37 ℃ for one hour. After three washes with PBS/0.2% Tween, 100. mu.l of standard curve was added. The standard curve concentration was incubated at room temperature for 2 hours. Next, plates were washed 3 times with PBS/0.2% tween. Next, 100. mu.l of the anti-hAPRIL.133 monoclonal antibody was added to the plate at a concentration of 1. mu.g/ml diluted in PBS/1% BSA and incubated at 37 ℃ for one hour. Subsequently, plates were washed three times with PBS/0.2% Tween and 100 μ L of goat anti-mouse IgG (H & L) (Southern Biotech, cat num 1031-05) diluted in PBS/1% BSA at 1:1,000 was added and incubated for one hour at 37 ℃. Plates were washed three times with PBS/tween 0.2% and addition of 100 μ l TMB substrate (TMB) allowed visualization of bound immune complexes. The reaction was stopped by adding an equal amount of 1M hydrochloric acid to the reaction volume. Binding to APRIL was quantified by measuring the OD at 450 nm. In this assay format using APRIL-binding peptides obtained according to the method of the invention, no effect was observed in the presence of human serum.

Claims (13)

1. An APRIL-binding immunoglobulin comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3 and 4, SEQ ID NO: 13 and 14, SEQ id no: 23 and 24, SEQ ID NO: 33 and 34, and SEQ ID NO: 43 and 44 of the amino acid sequence V_HAnd V_LA pair of domains.
2. 2.A cell comprising a nucleotide sequence encoding the APRIL-binding immunoglobulin of claim 1.
3. 3.A process for producing APRIL-binding immunoglobulins comprising: providing the cell of claim 2, culturing the cell, and allowing the cell to express and secrete the APRIL-binding immunoglobulin.
4. 4. APRIL obtained by the process of claim 3 binds to immunoglobulins.
5. 5. Use of the APRIL-binding immunoglobulin according to claim 1 or 4 in the manufacture of a reagent for use in an ex vivo diagnostic assay.
6. 6. The use of claim 5, wherein the diagnostic test is for diagnosing a condition selected from the group consisting of: cancer, a condition associated with inflammation, sepsis, allergy, autoimmune disease, or infection.
7. 7. The use according to claim 5, wherein in the assay APRIL-binding immunoglobulin is detected in a sample from a subject and the presence of APRIL-binding immunoglobulin is used as an indicator of the presence of APRIL.
8. 8. The use according to claim 5, wherein the diagnostic test is for detecting the amount of APRIL that forms a complex with the APRIL receptor equivalent administered to the subject, the test comprising:

   -providing APRIL-binding immunoglobulins immobilized on a solid support;

   -applying a sample from a subject to the solid support and incubating to allow complexes present in the sample to bind to the solid support;

   -washing;

   -detecting bound complexes and/or detecting bound uncomplexed APRIL.
9. An APRIL-binding immunoglobulin comprising SEQ ID NO: 3 and 4 amino acid sequence V_HAnd V_LA pair of domains.
10. An APRIL-binding immunoglobulin comprising SEQ ID NO: 13 and 14 amino acid sequence V_HAnd V_LA pair of domains.
11. An APRIL-binding immunoglobulin comprising SEQ ID NO: 23 and 24 of amino acid sequence V_HAnd V_LA pair of domains.
12. An APRIL-binding immunoglobulin comprising SEQ ID NO: 33 and 34 of amino acid sequence V_HAnd V_LA pair of domains.
13. An APRIL-binding immunoglobulin comprising SEQ ID NO: 43 and 44 of the amino acid sequence V_HAnd V_LA pair of domains.

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